The ability to induce therapeutically bone or cartilage formation would allow clinicians to treat a variety of congenital skeletal syndromes or accelerate bone fracture healing. As a model for such applications, craniofacial skeletogenesis involves the specification of cell fates in a tightly regulated spatiotemporal fashion. In this proposal, three sets of experiments will challenge the plasticity behind cell specification during development of the cranial skeleton, using both directed and unbiased approaches to identify and characterize factors that regulate skeletal cell fates in vivo. In both gain and loss of function approaches, embryonic cells that are known to give rise to specific tissue types (e.g., bone) in the zebrafish hyoid skeleton will be injected with compounds that modify expression of the skeletogenic transcription factors Sox9a, SoxQb, Runx2a, and Runx2b. Using transgenic methodologies combined with single cell clonal analyses, expression of these factors will be tightly correlated with ultimate cell fates. Finally, an unbiased genetic screen for zebrafish skeletal mutants will identify novel factors regulating skeletal cell specification. In total, this proposal will clarify at the cellular level the molecular mechanisms regulating skeletogenesis.